This invention relates to solid support reagents useful for synthesis. In recent years, solid phase synthesis has emerged as a powerful tool in high throughput synthesis for the preparation of small molecule libraries and oligomeric materials such as oligonucleotides, peptides, and carbohydrates. More particularly, this invention is directed to solid support reagents and methods of synthesizing oligonucleotides having a modified 3′-terminus.
Current methods to introduce chemical modifications into oligonucleotides employ special phosphoramidite reagents during solid phase synthesis. The primary aliphatic amine group is one of the most widely used chemical modifications since it reacts with many commercially available labeling reagents. Attention has focused on modification of the 5′ terminus and a number of protected amino-alkyl phosphoramidites have been reported to incorporate an amino group into the 5′ position of oligonucleotides. Agrawal, S., (1986) Nucl. Acids. Res. 14, 6115; Connolly, B. A., (1987), Nucl. Acids Res. 15, 3131; Jablonski, E., (1987) Nucl. Acids Res. 15, 5275; Smith, L. M., (1985) Nucl. Acids Res. 13, 2399; Connell, C., (1987) BioTechniques 5, 342; Sproat, B. S., (1987) Nucl. Acids Res. 15, 6181; Sinha, N. D. (1988) Nucleic Acids Res. 16, 2659. Oligonucleotides modified by these reagents can be subsequently derivatized with fluorophores, biotin, and other molecules.
It is also well-known that the usefulness of oligonucleotides can be enhanced by modification at the 3′-terminus e.g., by including small molecular weight groups at the 3′-end such as the 3′-tailed cholesterol oligonucleotides of Letsinger et al. used to inhibit HIV-1 replication (Proc. Natl. Acad. Sci. 86:6553-6556 (1989)) and 3′-tailed acridine oligonucleotides of Asseline et al. found to be stabilized intercalating agents which improved binding to complementary sequences (Proc. Natl. Acad. Sci. 81:8297-3301 (1984)). The stability of 3′-modified oligonucleotides in serum may also be enhanced. For example, unmodified oligonucleotides are rapidly degraded by 3′ exonucleases in serum containing media.
Techniques to prepare oligonucleotides with a modified 3′ terminus are less convenient and more tedious than the better known methods to prepare 5′ modified oligonucleotides. Thus, useful synthetic methods for the synthesis of 3′-substituted oligonucleotides have been slower to develop. Nelson et al. Nuc. Acids Res. 17:7179 (1989) has described a phosphoramidite reagent which he states can be incorporated at any position in the synthetic oligonucleotide. However, Nelson has only demonstrated the use of this reagent to incorporate a primary aliphatic amine on the 5′-terminus of an oligonucleotide sequence. Nelson et al. Nuc. Acids Res. 17:7187 (1989) has also described the synthesis of a multi-functional controlled pore glass (CPG) which can be used to incorporate 3′ terminal primary aliphatic amines into synthetic oligonucleotides. This reagent known as Amine-On CPG (Clontech) is commercially available but has been reported to give unpredictable results (See, Petrie, et al. Bioconj. Chem. 3:85 (1992)).
Petrie et al. Bioconj. Chem. 3:85 (1992) has reported an improved CPG support for the synthesis of 3′-amino substituted oligonucleotides. This support, 3′-aminohexyl CPG (AH-CPG), allows for the direct synthesis of oligonucleotides bearing a 3′-aminohexyl substituent. Petrie utilized the AH-CPG support to prepare an 11-mer oligonucleotide and showed it to be a distinct product by both polyacrylamide gel electrophoresis and reversed-phase HPLC. This AH-CPG method suffers from both slow cleavage from the solid support and amino group deprotection, which thereby limits the type of oligonucleotides that can be synthesized and often limits yield.
Reed et al. U.S. Pat. No. 5,419,966 has also reported a controlled pore glass matrix (CPG) support for oligonucleotide synthesis which has the following structure:
where 1,3-dioxoisoindoline-5-carboxamide serves as a linking group between the CPG support and the oligonucleotide chain. The wavy line represents a carbon chain which covalently links the NH group of the carboxamide with the controlled pore glass matrix, X is 2,2′-dimethyoxytrityl or H, and R is alkyl, aryl, arylalkyl, heteroalkyl, or heteroaryl. The dimethoxytrityl group is removed from the CPG support by treatment with acid, and the oligonucleotide is built, step-by-step, in a conventional synthesizer after attachment of the 3′ end of the first oligonucleotide unit to the hydroxyl function connected to the R group. Because methods to prepare oligonucleotides with a modified 3′ terminus are inconvenient and more tedious, useful synthetic methods for the synthesis of 3′-substituted oligonucleotides have been slow to develop. Accordingly, there is a clear need for improved methods to prepare oligonucleotides with a modified 3′ terminus.